RO122814B1 - PROCESS OF SEPARATING A MIXTURE OF 1,3-OXATIOLANE DERIVATIVES - Google Patents

Abstract

The invention relates to a process for separating a mixture of 1,3-oxathiolane derivatives, used for treating HIV infection. According to the invention, the process consists in subjecting the racemic mixture of 2-hydroxymethyl-5-(5-fluorocytosine-1-yl)-1.3-oxathiolane to a resolution stage, under the action of an enzyme selected from the group consisting of esterase, lypase, substilizine, alpha-chymotrypsin, citidine-deoxycitidine deaminase, which preferentially catalyzes a reaction of one of the enantiomers, followed by exposing the resulting product to the action of a second enzyme which enhances the resolution, recrystallizing the resolution product and treating the same with a chiral acid, chosen from a group consisting of malic acid, mandelic acid, 10-camphor sulfonic acid, 3-bromcamphor-8-sulfonic acid, di-benzoyl tartaric acid and di-para-toluoyl tartaric acid.

Description

Everyone has the right to make written and motivated submissions to

OSIM, a request for revocation of the patent, within 6 months from the publication of the mention of the decision granting it

RO 122814 Β1

The invention relates to a process for separating a mixture of 1,3oxathiolane derivatives used in the treatment of HIV infection.

It has been known since 1981 that acquired immunodeficiency syndrome (AIDS) is a disease that severely affects the human immune system and that almost without exception results in death. In 1983, the etiological cause of acquired immunodeficiency syndrome (AIDS) was determined to be the human immunodeficiency virus (HIV). in December 1990, the World Health Organization estimated that between 8 and 10 million people worldwide are infected with the human immunodeficiency virus (HIV), of which between 10,000 and 1400,000 are from the United States.

In 1985, synthetic nucleoside 3'-azido-3'-deoxythymidine (AZT) was reported to inhibit human immunodeficiency virus replication. Since then, other synthetic nucleosides have been shown to be effective against human immunodeficiency virus (HIV), including 2 ', 3'-didezoxyquinosine (DDI), 2', 3'-didezoxycytidine (DDC), 3'-fluoro-3 '-desoxytimidine (FLT) and 2', 3'-didezoxy2 ', 3'-didehydrotimidine (D4T).

It has also been shown that other 2 ', 3'-didezoxinucleosides inhibit the in vitro development of a variety of these viruses. It appears that, after cellular phosphorylation of 5'-triphosphate by cellular kinases, these synthetic nucleosides are incorporated into the developmental filament of viral deoxyribonucleic acid (DNA), thus causing the chain to stop, due to the absence of 3'-hydroxyl group.

The success of the various 2 ', 3'-didezoxinucleosides in inhibiting human HIV immunodeficiency virus replication in vivo or in vitro has led to a number of research topics for finding and testing nucleosides that substitute a heteroatom for the carbon atom at the 3' position of the nucleoside. Norbeck et al., Describe that (±) -1 - ((2p, 4P) -2- (hydroxymethyl) -4-dioxolanyl) thymine (refers to the form of (±) -dioxolane-T) exhibits modest activity compared to HIV immunodeficiency virus (EC ₅₀ of 20 pm in ATH 8 cells) and is not toxic to uninfected control cells at a concentration of 200 pM, Tetrahedron Letters 30 (46), 6246, (1989). European patent application WO / 0337713 and US patent 5041449, as IAF holder BioChem International, Inc., describe 1,3-dioxolans-2-substituted 4-substituted having antiviral activity.

US 5047407 and European patent application no. 0382526 also having the IAF holder BioChem International, Inc., describes a number of 1,3-substituted-5-substituted 1,3-oxathiolane nucleosides with antiviral activity, and specifically reports that the racemic mixture (approximately position C4 ' ) of the Cl'-β isomer of 2-hydroxymethyl-5- (cytosine-1-yl) -1,3oxathiolane (it refers further to (+) - BCH-189) has approximately the same activity as the immunodeficiency virus as AZT and is not toxic at the levels tested. It has also been found that (±) -BCH-189 inhibits AZT-resistant HIV immunodeficiency virus replication, which is isolated in vitro from patients who were treated with synthetic nucleoside 3'-azido-3'doxytimidine (AZT) for a long time. for more than 36 weeks.

Another virus that causes serious human health problems is the hepatitis B virus (HBV). Hepatitis B virus is inferior to tobacco only as a cause of human cancer. The mechanism by which the hepatitis B virus induces cancer is unknown, so it is postulated that it can directly trigger tumor development or may trigger tumor development indirectly through chronic inflammation, cirrhosis and cellular regeneration associated with infection.

After an incubation period of two to six months in which the host is infected with the hepatitis B virus, the disease progresses to the stage of acute hepatitis and to the destruction of the liver, which causes abdominal pain, jaundice and elevated levels of enzymes in the blood.

Hepatitis B virus can cause fulminant hepatitis, with rapid progression, often fatal as a form of disease, in which large portions of the liver are destroyed.

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Patients with acute hepatitis are often recovered. In some patients, however, 1 high levels of viral antigen persist in the blood for a long or indefinite period, causing a chronic infection. Chronic infections can lead to persistent chronic hepatitis. 3 Patients infected with persistent hepatitis B virus are mostly found in developed countries.

By mid-1991, there were approximately 225 million chronic hepatitis B virus carriers in Asia alone and nearly 300 million carriers worldwide. Persistent chronic hepatitis can cause exhaustion, liver cirrhosis and hepatocellular carcinoma, a primary liver cancer.

In western industrialized countries, groups at high risk for hepatitis B virus infection include those who come in contact with hepatitis B virus carriers or with blood samples. The epidemiology of hepatitis B virus is very similar 11 to that of acquired immunodeficiency syndrome, which explains why infection with hepatitis B virus is common among patients with AIDS syndrome or with complex 13 acquired immunodeficiency syndrome (AIDS). However, hepatitis B virus is much more contagious than human immunodeficiency virus (HIV). 15

A human serum-derived vaccine was developed to immunize patients against hepatitis B. While it has been found to be effective, vaccine production is 17 complicated, as the supply of human serum from chronic carriers is limited and the purification process is long and time-consuming. expensive. Moreover, each series of vaccines prepared 19 from different sera must be tested on chimpanzees for better safety. The vaccines were therefore produced by genetic engineering. Daily treatments with interferon alfa, a genetic engineering protein, have proven promising. However, for dating, there is no known pharmaceutical agent that effectively inhibits virus replication. 23 In order to market a nucleoside for pharmaceutical purposes, it must be not only effective, low in toxicity, but also cost-effective to manufacture. A major research and development theme has been oriented towards new processes, with low costs for large-scale nucleoside production. 2 ', 3'-27 didezoxynucleosides are currently prepared by one of the following two pathways: derivatizing an intact nucleoside or condensing a sugar fragment with a heterocyclic base. Although there are numerous disadvantages associated with obtaining new nucleoside analogues by modifying intact nucleosides, a major advantage of this process 31 is that the absolute absolute stereochemistry has already been naturally established. However, the process cannot be used in the production of nucleosides containing either bases that are 33 unnaturally produced or fragments of carbohydrates that are produced unnaturally (and which, for this reason, are not prepared from intact nucleosides). , such as, for example, 35 1,3-oxatiolan nucleosides and 1,3-dioxolan nucleosides.

If a carbohydrate fragment or a compound with a carbohydrate-like structure with a base is condensed to form a synthetic nucleoside, the nucleoside that is produced has two chiral centers (at positions C1 'and C4') and so on. there are 39 as diastereomeric pair. Each diastereomer exists as a set of enantiomers. Therefore, the product is a mixture of four enantiomers. 41

It has often been found that nucleosides with unnaturally produced stereochemistry either at position C1 or at position C4 'are less active than the same naturally occurring stereochemistry 43 nucleosides. For example, Carter et al. Reported that the concentration of (-) - carbovir enantiomer (2 ', 3'-didehydro-2', 3'-didezoxiguanosine) in cell culture 45 is required to reduce reverse transcriptase activity by 50% ( EC ₅₀ ) is 0.8 μΜ, while EC ₅₀ for (+) - carbovir enantiomer is greater than 60 μΜ. Antimicrobial 47

Agentsand Chemotherapy, 34: 6,1297-1300 (June 1990).

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WO 91/11186 discloses that 1,3-oxatiolan nucleosides may be prepared with higher diastereoselectivity (percentage of nucleoside superiors with configuration (3 at the link between the C1 'carbon atom and the heterocyclic base), by a careful selection of the Lewis acid used in condensation process It has also been found that condensation of the 1,3-oxathiolane nucleoside with a base occurs with almost complete β-stereospecificity, when tin chloride is used as a condensation catalyst. Other Lewis acids cause CT-selectivity β low (or zero) or cannot catalyze reactions.

Given the fact that acquired immunodeficiency syndrome, the AIDS complex and the hepatitis B virus have worldwide epidemic levels and have tragic effects on infected patients, there is a great need to provide new, effective pharmaceutical agents for the treatment of these diseases, which have a low toxicity to the host organism.

There is also a need to find a commercially viable, cost-effective process for producing pharmaceutically important nucleosides and achieving a β-stereospecificity at the C4 'position of synthetic nucleosides prepared by condensation. a fragment with a carbohydrate-like structure with a base.

The problem solved by the invention is to present a process for separating 1,3-oxathiolane derivatives.

The process for separating the 1,3-oxathiolane derivatives according to the invention removes the disadvantages above, in that the racemic mixture of 2 hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane is subjected to a resolution step below the action of an enzyme selected from the group consisting of esterase, lipase, substilizine, α-chemotrypsin, cytidindezoxycytidine deaminase, which preferably catalyzes a reaction in one of the enantiomers, followed by exposure of the resultant product to the action of a second enzyme selected from the esterase group, lipase α-chemotrypsin subtilisin, which enhances the resolution by promoting the binding reaction of one of the enantiomers, recrystallizing the resolution product and treating it with a chiral acid selected from a group consisting of malic acid, mandelic acid, 10-camphorsulfonic acid, 3- bromocamphor-8-sulfonic acid, di-benzoyl tartaric acid and di-paratoluoiltartric acid.

By applying the invention, the following advantages are obtained:

- 1,3-oxathiolane derivatives with increased antiviral activity are obtained;

- the present method is regioselective.

The process for preparing 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane consists of the reaction of 5-fluorocytosine, optionally protected with 1,3-oxathiolane of the formula:

wherein R _1a is a hydrogen atom or a hydroxy protecting group, an acyl group and L is a labile group; and, optionally, any hydroxy protecting group is removed.

Another process for the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane is the reaction of a compound of the following formula:

NHRt _b

S

RO 122814 Β1 wherein R _1a is a protecting group of the hydroxy group and R _1b is a protecting group of the amino group, with a fluorination agent which introduces the fluorine atom into position 5 of the cytosine cycle and then, optionally, is removed protection groups.

Another process for the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane is the reaction of a compound of the formula:

wherein R _1a is a hydroxy protecting group, with an agent which converts the oxo group to position 4 of the uracil ring in the amino group and then the protecting groups are removed.

The 1,3-oxathiolane derivative is prepared by: (i) ozonating the compound of the formula CH ₂ = CH-CH ₂ -OR or ROCH ₂ -CH = CH-CH ₂ OR, wherein the radical R is a protecting group, obtaining the compound of formula: 19

(ii) reacting the compound obtained in step (I) with mercaptoacetic acid to form the compound with the structure:

(iii) reducing the compound obtained in step (ii) and then reacting it with the carboxylic acid anhydride, with an alkylating agent or halogenating agent, to obtain the compound of the formula:

wherein L is a labile group.

Another object of the present invention is the pharmaceutical composition comprising as active compound a 1,3-oxathiolane derivative and a carrier for oral administration and is conditioned in capsule or tablet form.

Thus, an object of the present invention is a method and composition for the treatment of human patients infected with the human immunodeficiency virus, HIV.

Another object of the present invention is to provide a method and composition for the treatment of human patients or other host animals infected with hepatitis B virus, HBV.

Another object of the present invention is also enantiomerically enriched nucleosides in 1,3-oxathiol.

The present invention further contains a method of decomposition of C4 'enantiomers of 1,3-oxatiolan nucleosides.

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For the treatment of infections with the human immunodeficiency virus HIV and the hepatitis B virus, HBV, in humans and other host animals, according to the present invention, for the treatment mentioned above, an effective amount of 2-hydroxy methyl 5- (5-) is provided. fluorocytosin-1-yl) -1,3-oxathiolane, a derivative of this pharmaceutically acceptable compound, including 5 'or N ⁴ alkylated derivative or 5' or N ⁴ acylated derivative or a salt of this acceptable compound pharmaceutically acceptable, in a pharmaceutically acceptable vehicle.

Also, it was found that 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC) surprisingly exhibits intense activity against human immunodeficiency virus, with very low toxicity to the host cell. It has also been found that the compound 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC) exhibits very significant activity against hepatitis B virus, HBV, and therefore may be used for the treatment of patients who have a variety of diseases associated with hepatitis B virus infection, HBV.

Toxicity and pharmacokinetic studies confirm the complete use of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxatiolan (FTC) as an antiviral agent for pharmaceutical administration. The FTC compound and its enantiomers are non-toxic to human bone marrow cells, peripheral, at concentrations over 50 μΜ and other cell lines at concentrations over 200 μΜ. The FTC-TP compound is a major intracellular metabolite in PBMC and HepG2 cells. The FTC-TP compound competitively inhibits human HIV-1 immunodeficiency virus as reverse transcriptase (RT) with K1 for 0.2 µM, using a template-primer poly (l) oligo (dC). Using sequential analysis, the FTC-TP compound is shown to be a potent terminator of the DNA deoxyribonucleic acid chain, when using the human HIV-RT immunodeficiency virus (C-stops).

Chronic treatment with FTC is not toxic to rodents, even at oral doses of 85 mg per kilogram for one day for at least two months. Pharmacokinetic studies of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC) in Rhesus monkeys indicate high oral bioavailability (approximately 73 + 6%) and a terminal half-life about 1.34 + 0.18 (mean oral and intravenous administration).

Also described herein is a process for decomposing a racemic mixture of nucleoside enantiomers, including the racemic mixture of FTC, which includes the phase of exposure of the racemic mixture to the action of an enzyme that preferentially catalyses a reaction in one of the enantiomers. . The process can be used to resolve a wide range of nucleosides, including pyrimidine nucleosides and purine nucleosides, which are optionally substituted for the carbohydrate or the base portion. The process can therefore be used to resolve nucleoside derivatives containing additional heteroatoms in the carbohydrate moiety, for example (±) -FTC and (±) -BCH-189. Nucleoside resolution can be achieved widely at moderate costs. Using the models described in the present invention, the FTC can be separated into its (+) - beta-D and (-) - beta-L enantiomers. The (-) - beta-L enantiomer appears to be much more active than the (+) - beta-D enantiomer to HIV, HBV and SIV. The (+) enatiomer of the FTC is therefore active against HIV, HBV and HIV.

As used in the present invention, the term "enantiomerically enriched nucleoside" refers to a nucleoside composition, including at least 95% of a single enantiomer of that nucleoside.

As used in the present invention, the term "FTC" refers to the compound

2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (racemic form or enantiomers), therefore to the 2'-deoxy-5-fluoro-3'-thiacitidine compound.

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As used in the present invention, the term (±) -FTC refers to (±) -beta-D, L-2-h methoxy-5- (5-fluoro orocytosin-1-yl) -1, 3-oxathiolane.

As used in the present invention, the term (-) - FTC refers to (-) - beta-L-2-hydroxymethyl-beta-5- (5-fluorocytosin-1-yl) -1,3-oxathiol.

As used in the present invention, the term (+) - FTC refers to (+) - beta-D2-hydroxy methyl-5- (5-fl uorocytosin-1-yl) -1,3-oxathiol.

As used herein, the terms FTC-MP, FTC-DP and FTC-TP refer to FTC monophosphate, FTC diphosphate and FTC triphosphate respectively.

As used herein, the term BCH-189 refers to 2-hydroxymethyl-5- (5-cytosin-1-yl) -1,3-oxathiolane.

As used in the present invention, the term preferential enzyme catalysis refers to catalysis using an enzyme of one substrate instead of another substrate.

As used in the present invention, a mobile group is a functional group that forms at an incipient carbonation when separated from the molecule to which this group is bound.

In conclusion, a process for separating a mixture of 1,3-oxathiolane derivatives is presented in the present invention, and the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane consists of:

(i) the reaction between 5-fluorocytosine, optionally protected, with 1,3-oxathiolane having the formula wherein R _1a is hydrogen or a protecting group of the hydroxyl group, including an acyl group and L is a mobile group; and, optionally, any hydroxyl group protection group is removed;

(ii) reaction of the compound of formula B:

(wherein R _1a is defined as above and R _1b is a protecting group of the amino group), with a fluorination agent participating in the introduction of a fluorine atom at position 5 of the cytosine ring; or (iii) the reaction of a compound of formula C:

RO 122814 Β1 (wherein the radical R _1a is defined as above), with an agent participating in the transformation of the oxo group at position 4 of the uracil ring into amino group; any remaining protecting group may be removed to obtain the desired compound;

In the process, in step (i), the hydroxy group protecting group includes the protecting groups described in detail below, including acyl (e.g. acetyl), arylacyl (e.g., benzoyl or substituted benzoyl), trityl or monomethoxytrityl, benzyl groups. or substituted benzyl, trisubstituted silyl including trialkylsilyl (eg, dimethyl-t-butylsilyl) or diphenylmethylsilyl. The 5-fluorocytosine compound may optionally be protected with trisubstituted silyl groups. Protection groups can be removed by any conventional method. Mobile group L is a typical mobile group, known in the literature, for nucleoside chemistry, for example, halogen such as chlorine or bromine, alkoxy such as methoxy or ethoxy or acyl such as, for example, acetyl or benzoyl.

The reaction (s) may be carried out in an organic solvent (eg, 1,2-dichloroethane or acetonitrile) in the presence of a Lewis acid, preferably tin chloride or trimethylsilyl triftalate.

Compounds of formula A (wherein L represents an acyl group, for example, an acetyl group) can be obtained by reacting the compound of formula D:

(where R _1a is defined as above) with a reducing agent, for example aluminum lithium hydride, and then treated with a suitable conventional reagent to obtain the desired intermediate compound, for example carboxylic acid anhydride. , such as acetic anhydride, for acylation, chlorination or bromination reagents for halogenation or alkylating reagents.

The compound of formula D can be prepared by reacting the compound of formula E:

H ^Β ι. ° ^ν - ^Ζ ^ »ο ^E with compound HSCH ₂ CO ₂ H at a high temperature.

The compound of formula E may be prepared by ozonolysis, starting from an allyl ether or allyl ester of formula CH ₂ = C-CH ₂ -OR or a dieter or diester of 2-butene-1,3-diol of formula RO- CH ₂ -CH = CH-CH ₂ -OR, wherein R is a protecting group, such as, for example, an alkyl, silyl or acyl group.

Regarding step (ii) of the process, the 5-fluorine substituent can be introduced by methods known in the literature (MJ Robins et al., In Nucleic Acid Chemistry, Part 2, LB Townsend and RS Tipson, editors, J. Wiley and Sons, New York, 895-900 (19/8) and references included; R. Duschinsky in Nucleic Acid Chemistry, Part 1, LB Townsend and RS Tipson, editors, J. Wiley and Sons, New York43-46 (1978) and the references included). The fluorinating agent may, for example, be trimethylhypofluoride in fluortriclormethane.

With respect to step (iii) of the process, the compound of formula C may be treated with 1,2,4-triazole, together with 4-chlorophenyl dichlorphosphate, to form the corresponding 4- (1,2,4-triazooyl) compound, which is then converted to the desired 4-amino compound (cytidine), for example by reaction with methanol.

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The starting materials of formulas B and C may be prepared, for example, by reacting a corresponding (optionally protected) base 1 with a compound of formula A in a manner similar to that described in step (i). 5-Fluorourac and 5-fluor-3 ocitozine are commercial compounds provided by Aldrich Chemical Co., Milwaukee, Wl 53233, USA. 5

The resolution of the enantiomers (+) can be completed as will be specified in detail below. 7

The FTC compound can be converted to the pharmaceutically acceptable ester by reaction with a suitable esterification agent, for example, an acid halide 9 or an anhydride. The FTC compound or the pharmaceutically acceptable derivative of this compound can be converted into a pharmaceutically acceptable salt, 11 in a convenient manner, for example, by treating it with a suitable base. The ester or salt of the FTC can be converted to the FTC, for example by hydrolysis. 13

I. Preparation of active compounds

The racemic mixture of the FTC compound can be prepared according to the method described in detail in PCT application publication WO 91/11186, published on August 8, 1991, as Emory University holder, or by the process described in Example 1. In general, the method includes 17 ozonation of either an allyl ether or allyl ester of the formula CH ₂ = CH-CH ₂ -OR or of a dieter or diester of 2-butene-1,3-diol of the formula RO-CH ₂ -CH = CH-CH ₂ -OR, wherein the radical R is a protecting group, such as alkyl, silyl or an acyl group, to form a glycaldehyde of the formula OHC-CH ₂ -OR; adding thioglycolic acid 21 to glycaldehyde to form a lactone of formula 2- (R-oxy) -methyl-5-oxo-1,3oxathiolane; reducing lactone to different compounds containing a mobile group in position 5 of the 23 oxathiolane cycle; coupling of these compounds with 5-fluorocytosine-silyl in the presence of SnCl ₄ to form the beta isomer of FTC; and optional removal of protection groups. 25

The following are examples of embodiments of the invention, in connection with FIG. 1 ... 12, which represents: 27

FIG. 1 is an illustration of the chemical structure of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC);

FIG. 2, an illustration of the process for the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane;

FIG. 3, a flow diagram of the alkaline phosphatase specificity and the phosphodiesterase in snake venom, for the (+) and (-) enantiomers of the FTC compound; 33

FIG. 4, a graph showing the evolution of lipase-catalyzed hydrolysis, of the 5'-butyryl ester of the FTC compound over time, using the enzymes Amano PS-800® (open square) and PLE 35 (open open circle);

FIG. 5, a graph of the effect of the concentration (μΜ) of the racemic compounds and enantiomers 37 of the enriched FTC (prepared by the method of example 4) against the percentage inhibition of human PBM cells infected with HIV-1 (closed circle, (±) -FTC), (open circle, (-) - FTC), 39 (closed surface, (+) - FTC);

FIG. 6, a graph of the concentration of FTC compound (μΜ) enriched racemic and enantiomeric (41 prepared by the process of example 3) on the percentage inhibition of cells

Human PBM infected with ΗIV-1 (closed, (±) -FTC), (open circle, (-) - FTC), (closed square, 43 (+) - FTC);

FIG. 7, a graph of absorption of tritium saturated (+) - FTC compound in human PBM 45 cells (mean of two determinations) in time (hours) compared to pmol / 10 ⁶ cells;

FIG. 8, a graph of the output of the radiolabeled (±) -FTC compound from human PBM cells, 47 measured in hours relative to pmol / 10 ⁶ cells;

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FIG. 9, presence of compound [ ³ H] (±) -FTC and its phosphorylated derivatives in human HepG-2 cells (mean of two determinations), incubated in a medium containing 10 μΜ [ ³ H] (±) -FTC , measured in pmol / 10 ⁶ cells over time;

FIG. 10, the output of the compound [ ³ H] (±) -FTC and its phosphorylated derivatives in human HepG-2 cells in pmol / 10 ⁶ cells versus cells exceeded as time after cells were pulsed by 10 μΜ [ ³ H] (±) -FTC (700 DPM / pmol) for 24 h and evaluating the concentration of the compound at 24 h after elimination;

FIG. 11, decrease in the combined concentration of [ ³ H] (±) -FTC and its phosphorylated derivatives in human HepG-2 cells after incubation with 10 μΜ [ ³ H] (±) -FTC (700 DPM / pmol) for 24 h, in pmol / 10 ⁶ cells outdated as time;

FIG. 12, a graph of the effect of FTC enantiomers on the formation of granulocyte macrophage precursor cell colony, as measured, in percent survival versus concentration in μΜ (-) - FTC, open circle; (+) - FTC closed circle; AZT, closed square.

Example 1. Preparation of (±) -beta-D _J L-2-hydroxymethyl-5- (5-fluorocytosin-1yl) -1,3-oxathiolane

The process for preparing the racemic mixture of the FTC compound is illustrated in FIG. 2 and is described in detail below.

Protection of 2-butene-1,4-diol in a 2 L vial, with 3 necks, dry, in inert atmosphere, 100 g (93.5 ml = 1.135 moles = 1.00 equivalents) 2-buten-1, 4-diol and 15 g (about 0.1 equivalents) of DMAP (4-dimethylaminopyride) are dissolved in 800 ml of dried pyridine and kept under stirring, while the reaction mixture is cooled to 0 ° C. Then, butyryl chloride (260 ml = 2.2 equivalents) is added slowly to prevent overheating and the mixture is left stirring for one hour. The reaction is terminated by adding small amounts of ice water to the oil. The liquid is then decanted from the salt and evaporated in vacuo. The remaining salt is dissolved in water and the aqueous solution is extracted twice with ethyl ether. The other combined layers are washed once with a saturated solution of CuSO ₄ , twice with a saturated solution of NaHCO ₃ containing Norit® and then filtered under vacuum through a celite® filter.

The concentrated reaction mixture is dissolved in ether and washed, following the same procedure as above for the salt solution. The combined organic layers are concentrated on the rotary evaporator and then brought under vacuum. In order for this reaction to proceed quantitatively, the closed environment is required. The vacuum can be increased as needed. The product, 1,4-dibutyryl-2-butene-1,4-diol, is colorless to slightly yellow, in the form of a clear liquid.

Ozonolysis of protected diol

The 1,4-dibutyryl-2-butene-1,4-diol compound (1,365 moles) is dissolved in 41 methylene chloride (CH ₂ Cl ₂ ), dried in a 51-neck dry vial of 51 capacity. equipped with a large drying tube and an open gas inlet tube. The tube is an unfriteed gas bubbling tube that will be sealed by exposure to the concentrated solution. The solution is then stirred and cooled to -70 ° C, while inert gas is bubbled through the solution. The gas inlet is sealed, while the solution is sufficiently cooled and the vial and shaker are transferred to the ozone generator. The oxygen is bubbled through the solution kept under stirring for at least 20 minutes while it is kept on an ice bath. A Cryocool appliance is ideal for maintaining a low temperature for a long lasting reaction. The ozone is then introduced at 0.56 kg / cm ² 0.60 kg / cm ² (8 to 8.5 psi). After completion, the ozone flow is stopped and the oxygen is bubbled through the solution for about half an hour before adding 3 equivalents of Me ₂ S. The vial is then removed from the ice bath and transported to a niche, where it is kept under stirring for about 2 days, to obtain a complete reduction. The solution is then evaporated and brought under vacuum for several hours.

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This reaction typically produces about 95% protected aldehyde (2-1 butyryloxyacetaldehyde), a clear, colorless to yellow liquid.

Cyclization of aldehyde with mercaptoacetic acid 3

The aldehyde (1.0 equivalents) is dissolved in toluene to obtain a solution of 0.80 to 0.85 M, which is brought into a vial fitted with a Dean 5 Stark separator. Thioglycolic acid (1.1 equivalents) is added and the mixture is heated to reflux temperature. The water is azeotropically removed using the separator. The reaction is complete within 7 3 hours and the mixture is kept cold at room temperature. The organic solution is washed twice with equal volumes of saturated aqueous sodium bicarbonate NaHCO ₃ and 9 and then washed once with water, dried over MgSO ₄ and Norit magnesium sulfate, then filtered under vacuum through celite before to be evaporated in vacuo. The first aqueous NaHCO ₃ sodium bicarbonate solution is extracted once with ether; the ether is washed once with water, dried over MgSO ₄ and Norit® magnesium sulphate, filtered through celite® under vacuum and evaporated to 13 other organic materials from the toluene solution. The combined material is placed under vacuum until the next day. Typically, the reaction yields a 90% yield of 2- (butyryloxy) methyl-5-oxo-1,3-oxathiolane. 17

Reduction of lactone and conversion to acetate

The 2-butyryloxy-methyl-5-oxo-1,3-oxathiolane compound (1.00 equivalent) is dissolved in dried tetra-19 hydrofuran (THF) to give a 0.23 M solution which is brought to a dry bottle provided with 3 necks and equipped with a mechanical shaker and kept in an inert 21 atmosphere. The solution is stirred and cooled to 0 ° C, after an equivalent of 1.0 M Li (t-BuO) ₃ AIH in tetrahydrofuran (THF) was added by cannula. The reduction is 23 complete in about three hours, as observed by TLC thin-layer chromatography, using as solvent system, 2 parts ether and 1 part hexane and anisaldehyde as 25 dyes.

About 10 equivalents of Ac ₂ A fresh distillate is then added and kept under 27 stirring for 2 days to obtain the acetylated product. The reaction is completed by adding the saturated NaHCO ₃ sodium bicarbonate solution, which is stirred for up to 29 days. The solution is then evaporated and stirred with more NaHCO ₃ sodium bicarbonate solution until the next day. This solution is then extracted with ether which is then washed (carefully) twice with saturated NaHCO ₃ sodium bicarbonate solution and once with water, then dried over MgSO ₄ and Norit® magnesium sulfate, and then filter through 33 celite® under vacuum and evaporate. The product is a clear, dark yellow liquid. Gas-chromatographic analysis (Initial temperature -80 ° C; Initial time = 5 min; Progression rate - 35 10 / min; Final temperature = 240'C) indicates a typical purity of about 70%.

Silylation of 5-fluorocytosine compound 37

5-fluorocytosine (1.05 equivalents based on the amount of acetylated lactol obtained in the previous phase, using the gas chromatographic method to indicate purity) is silylated 39 by refluxing at least 10 equivalents of hexamethyldisilazane containing a catalytic amount of ammonium sulphate pure (0.05 to 0.10 equivalents) for two hours, after which solution 41 becomes clear. The vial is then sealed tightly and the solvent is removed, using a vacuum pump with an auxiliary separator. The product, in white solid form, is left in vacuo until the next day, until ready for use in the next coupling reaction phase. 45

Coupling of the silylated 5-fluorocytosine compound with acetylated lactol

The silylated 5-fluorocytosine compound (33.86 g, 0.124 mol), in dry dichloromethane (350 ml), 47 forms a solution to which is added a solution of SnCl ₄ tin tetrachloride (135.6 ml o

EN 122814 Β1 1 molar solution in methylene chloride CH ₂ Cl2) under nitrogen atmosphere. The solution is stirred for 15 minutes at room temperature. This solution is added to the solution of lactol acetate through a thin tube (38 g, 0.113 mol) in dichloromethane (400 ml) under a nitrogen atmosphere for over 30 min.

The reaction solution is stirred for 2 h, at which point the reaction is complete, as indicated by thin layer chromatographic analysis. The reaction solution is then diluted with dichloromethane (500 ml) and the reaction is stopped upon addition of the ammonium hydroxide solution. The ammonium hydroxide solution (100 ml) is added slowly, maintaining the reaction temperature below 30 ° C, resulting in the formation of a white precipitate. The mixture is then stirred for a further 30 minutes and then passed through a column in the form of a silica gel plug (7 inches 17.78 cm in diameter and 2 inches 5.08 in height). The column is sequentially eluted with dichloromethane (2 I), ethyl acetate (2 I) and a mixture of 9 parts ethyl acetate and 1 part ethanol (4 I). The eluent ethyl acetate and the mixture of ethyl acetate and ethanol contain the desired product. These solutions are combined and evaporated under reduced pressure. The residual sticky solid product is then washed with dry ether (20 ml) to give a white solid product (25.35 g; 71%) of FTC-5'-butyrate.

The FTC-5'-butyrate product (8.74 g; 0.026 mol) is dissolved in 250 ml of methanol. Sodium methoxide (2.85 g; 0.052 g) was added at room temperature. The reaction mixture is stirred for 1 h, at which point the reaction is complete, as confirmed by TLC thin-layer chromatographic analysis. The NH ₄ Cl ammonium chloride solution (10 ml) is added to stop the reaction and then the solvent is removed under reduced pressure. The residue is then absorbed on silica gel (5 g) and passed through a narrow column, using as a mixture a mixture of 9 parts ethyl acetate and 1 part ethanol. The fractions containing the product are combined and evaporated, when a sticky solid product is washed which is washed with dry ether, when a white FTC solid product is formed (6 g, 88%).

The nuclear magnetic resonance analysis has the following results:

( ¹ H NMR: (DMSO-dg) 8.18 (1H, d, H _e , J = 8.4 Hz), 7.81 & 7.57 (2H, extended, NH ₂ ), 6.12 (1H , dd, H _1f J = 5.7 & 4.2 Hz), 5.40 (1H, t, OH, J = 5.7 Hz), 5.17 (1H, t, 1H ₄ , J = 3-6 Hz), 3.74 (2H, m, 2H ₅ ), 3.41 (1H, dd, 1H ₂ , J = 5.7 & 11.7 Hz), 3.11 (1H, dd, 1H ₂ , J = 4.2% (11.7 Hz);

¹³ C NMR: (DMSO-d _B ) 157.85 (d, J = 13.4 Hz), 153.28,136.12 (d, J = 241 Hz), 126.01 (d, J = 32.6 Hz ), 86, 90, 86, 84, 62, 48, 37, 07; mp 195-196 ° C.

II. Resolution of nucleoside enantiomers

A method is presented in the present invention for the resolution of racemic mixtures of nucleoside enantiomers, including but not limited to FTC (+) and (-) enantiomers. The method can therefore be used to separate racemic carbohydrate mixtures or carbohydrate-like portions, such as arsenic, for example, 1,3-oxatiolane and 1,3-dioxolane derivatives. The method consists in using an enzyme which preferably catalyzes a reaction of an enantiomer in a racemic mixture. The reacted enantiomer is separated from the unreacted enantiomer based on a new difference in physical structure. By the description given in the literature, it is possible to choose an enzyme that is selective for the nucleoside enantiomer of choice (or selective for the unwanted enantiomer, as a method of eliminating it), by selecting one of the enzymes that will be discussed further. down or by systematic evaluation of other known enzymes. From the specialized literature, it is thus known how to modify the substrate as much as necessary, in order to reach the desired resolution. By using displacement agents by chiral nuclear magnetic resonance, polarimetry or liquid chromatography

EN 122814 Β1 of high chiral performance, the optical ennoblement of the separated ester can be determined 1 The following examples illustrate the use of enzymes for the separation of racemic mixtures of the enantiomers. Other known methods of resolving racemic mixtures 3 may be used in combination with the resolution method described in the present invention.

All of these modifications are considered to be part of the scope of this invention. 5

Resolution based on hydrolysis of C5 'nucleoside esters

The method includes the reaction of the C5'-hydroxyl group of the racemate mixture of nucleoside 7 with an acyl compound to form the C5 'ester wherein the nucleoside is at the carbinol end of the ester. The racemic mixture of nucleoside esters 05 'is then treated with the enzyme which 9 preferably cuts or hydrolyzes one of the enantiomers and not the other, over a given period of time. 11

An advantage of this method is that the method can be used for the separation of a wide variety of nucleosides, including pyrimidine nucleoside and purine nucleoside, which 13 are optionally substituted in the carbohydrate or base portion. The method can therefore be used to separate nucleoside derivatives containing additional heteroatoms 15 in the carbohydrate moiety, for example FTC and BCH-189. The broad applicability of these methods lies in the fact that, although the carbinol portion of the ester plays a role 17 in the ability of an enzyme to differentiate the enantiomers, the major recognition portion for these enzymes is the carboxylic acid portion of the ester. Moreover, one can successfully extrapolate the result obtained from one enzyme to the study on the substrate of another enzyme in apparently different system, ensuring that the carboxylic acid portions of the two 21 substrates are the same or substantially similar.

Another advantage of this method is that it is regioselective. 23 Enzymes that typically hydrolyze esters do not catalyze foreign reactions in other portions of the molecule. For example, the lipase enzyme catalyzes the hydrolysis of 2-hydroxymethyl-5-25 oxo-1,3-oxathiolane ester without hydrolyzing the internal lactone. This contrasts markedly with the chemical methods of ester hydrolysis. Another advantage of this method is that the separation of the non-hydrolyzed enantiomer and the hydrolyzed enantiomer from the reaction mixture is very simple. The non-hydrolyzed enantiomer 29 is more lipophilic than the hydrolyzed enantiomer and can be efficiently recovered by simple extraction with one of a wide variety of non-polar organic solvents or from mixtures of 31 solvents, including hexane and the mixture of hexane and ester. The hydrolysed enantiomer, more polar, least lipophilic, can then be obtained by extraction with much more polar organic solvents, for example, ethyl acetate, or by lyophilization, followed by extraction with ethanol or methanol. Alcohol should be avoided during hydrolysis, as it can distort enzymes under certain conditions.

Enzymes and substrates 37

With a suitable combination of enzyme and substrate, conditions can be set for isolation of any nucleoside enantiomer. The desired enantiomer can be isolated by treatment of the racemic mixture with an enzyme that hydrolyzes the desired enantiomer (after which extraction of polar hydrolysate with polar solvent) or by treatment with an enzyme which hydrolyzes the unwanted enantiomer (after which the unwanted enantiomer is removed with a nonpolar solvent ). 43

Enzymes that catalyze the hydrolysis of esters include esterases, for example, pig liver esterase, lipases including swine pancreatic lipase and Amano PS-Boo, lipase, 45 substillisin and alpha-chemotrypsin.

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Fig. 3 is the flow diagram of the specificity of the alkaline phosphatase and phosphodiesterase from snake venom for the (+) and (-) enantiomers of the FTC compound. As indicated, alkaline phosphatase hydrolyzes the triphosphate of both enantiomers to the FTC compound and is therefore ineffective as a means of separation. Phosphodiesterase I preferentially hydrolyzes the (+) isomer of the FTC compound in its monoester, which can then be exposed to 5'-nucleotidase to obtain the (+) -FTC compound.

The most effective acyl group for esterification at the C5 'position of the nucleoside can be determined without inappropriate experimentation, by evaluating a number of homologues, using a selected enzyme system. For example, when 1,3-oxatiolan nucleosides are esterified with butyric acid, resolutions with both pork liver esterase and Amano PS-800 esterase, higher enantioselective activity (94-100% enantiomeric excess) and selectivity are performed. opposite. Esterase in pig liver preferentially hydrolyzes the (+) enantiomer of the FTC compound and the Amano-800C enzyme preferentially hydrolyzes the (-) enantiomer of the FTC compound. The percentage of enantiomeric excess reported in Table 1 is the amount of purified butyrate ester, which remains in the enzymatically treated mixture (for example, the enantiomer (-) butyric ester of the FTC in pig liver esterase and the enantiomer (+) butyric ester. of the FTC compound in the case of Amano PS-800).

Unlimited examples of acyl groups that can be evaluated for use with an enantiomeric mixture of nucleosides and in particular enzymes, include substituted alkyl carboxylic acids and alkylated carboxylic acids, including acetic acid, propionic acid, butyric acid and pentanoic acid. Together with certain enzymes, it is preferred to use an acyl compound which is significantly free of free electrons to facilitate hydrolysis by weakening the ester bond. Examples of acyl groups not significantly free of free electrons include alpha-halogens, such as 2-chloropropionic acid, 2-chlorobutyric acid and 2-chloropentanoic acid. Alpha-halogens are excellent substrates for lipases.

Resolution conditions

Enzymatic hydrolyses are typically performed with a catalytic amount of enzyme in an aqueous buffer solution having the optimum pH close to the enzyme discussed. As the reaction proceeds, the pH values result from the release of carboxylic acid. The aqueous base will be added to maintain the pH value close to the optimal value for the enzyme. The reaction process can be easily determined by monitoring the pH change and by the basic amount needed to maintain the pH. The hydrophobic ester (the non-hydrolyzed enantiomer) and the much more polar alcohol (the hydrolyzed enantiomer can be sequentially and selectively extracted from the solution by a judicious choice of organic solvents. Alternatively, the material to be separated can be passed through a column containing the enzyme. immobilized on a solid support Enzymatic hydrolyses performed under heterogeneous conditions may suffer from reduced reproducibility. Therefore, hydrolysis is preferable to be carried out under homogeneous conditions. Alcoholic solvents are not preferred, as they may distort the enzymes. by using non-ionic surfactants, such as Triton X-100. However, the addition of these surfactants not only helps dissolve the starting material, but also contributes to the solubility in water of the product. it can occur much more efficiently by adding a t agent non-ionic surfactant than under heterogeneous conditions, isolating both the raw material and the resulting product can be made much more difficult. The product can be isolated by much more convenient chemical and chromatographic processes (for example, selective salt formation). Diacylated nucleosides can be used, but they are often very lipophilic and strong, so they can be dissolved in the environment used.

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Example 2. Enantioselective hydrolysis of FTC compound catalyzed by lipase 1

A number of 5'-O-acyl derivatives of the FTC compound are prepared by selective O-acylation of the N-hydrochloride salt (see Table 1 and Fig. 4) of the (±) -FTC compound. The efficiency of hydrolysis of derivatives by lipases was investigated. As shown in Table 1, pig liver esterase (PLE) exhibits a higher level of selectivity for hydrolysis to the enantiomeric ester (+) of the FTC compound, by predominantly eliminating the butyrate enantiomer (-) of the FTC compound in the mixture. analyzed by high performance liquid chromatography. 7 By contrast, the enzyme PS-800 hydrolyzes the ester of the (-) enantiomer of the preferential FTC, by predominantly eliminating the butyrate of the enantiomer (+) of the FTC 9 in the mixture analyzed by high performance liquid chromatography. The degree of hydrolysis was found to be dependent on the nature of the acyl group; the acetyl derivative is significantly more inactive than the butyryl derivative. It has now been found that the degree of hydrolysis of propionic acid ester of the FTC is even faster than that observed for the butyrate derivative. 13

The percentage of separation and the percentage of enantiomeric excess were determined, both using high performance HPLC liquid chromatography. Thus, enantioselectivity is excellent when using pig liver esterase (PLE) (typically 97% ee, or higher), additional enrichment may be accompanied by 17 sequential enzymatic hydrolysis reactions, in which enantiomeric butyrate is enriched of enzyme-catalyzed hydrolysis of PLE pig liver is subjected to enzymatic hydrolysis with PS-800. 19

Table 1

Comparison of the effect of ester on enzymatic hydrolysis

substrate %Separation % E.e. (M.S.) FTC Esters with PLE: (-) - FTC (butyrate) Acetate 32.68 N.D. propionate 39.87 N.D. butyrate 48.00 98 butyrate 45.71 98.6 FTC Esters with PS-800: (+) - FTC (butyrate) Acetate 73.17 N.D. propionate 52.67 N.D. butyrate 58.34 N.D. butyrate 41,50 94

Example 3. Process for the preparation of (+) - and (-) - FTC by enantioselective hydrolysis 37 of FTC butyrate, catalyzed by lipase

The 5'-O-butyrate of the (±) -FTC compound (0.47 mmol, 149 mg) was dissolved in 16 ml of solution 39 consisting of 4 parts buffer solution and 1 part CH ₃ CN at pH = 8. The clear solution is shaken and treated with 26 mg of pork liver esterase (PLE-A). The evolution of the reaction is monitored 41 by high performance liquid chromatography (HPLC) (fig. 4). After 20 h (52% conversion), the reaction mixture is extracted twice with 80 ml of CHCl ₃ chloroform and then with 43 ml of ethyl acetate. The organic layer of the extracts is combined, dried over anhydrous magnesium sulfate MgSO ₄ , then filtered and concentrated by rotary evaporation. Residue 45

Thus, the result is eluted on 2x1000 m pTLC disks, using ethyl acetate as the eluent (double elution), to obtain after isolation 53 mg (36% based on the starting material) of FTC butter, which is determined by HPLC high performance liquid chromatographic analysis as having an enantiomeric excess of 98%. Enantiomerically enriched butyrate is then treated with 1.6 ml of methanol and then with 0.38 mmol (20 mg) of sodium methoxide. The resulting mixture is stirred at room temperature and the evolution of the reaction is monitored by high performance liquid chromatography, HPLC. The reaction is complete in 30 min. The solvent is evaporated off by rotary evaporation to give a white solid (76 mg) crude product, which is eluted on 1000 m pTLC, using as eluent a mixture of 5 parts of ethyl acetate and 1 part of ethanol. The (-) enantiomer of the FTC is isolated as a white solid product (33 mg; 82% yield). High-performance HPLC chromatographic analysis of FTC as a 5'-O-acetate derivative shows 97% enantiomeric excess (ee): [oc] ( ²⁰ , D) -120.5 ° (c = 0.88; in absolute ethanol) . The emulsions in the processing phase should be avoided by the addition of HCCI ₃ chloroform to the reaction mixture for completion (which therefore serves to denature the enzyme), followed by stripping the solvents under vacuum and then extracting with HCCI ₃ chloroform.

Similarly, 1.2 mmol (375 mg) of 5'-0-butyrate of (±) -FTC is dissolved in 40 ml of mixture consisting of 4 parts buffer solution pH = 8 and 1 part CH ₃ CN. The clear solution is shaken and treated with 58 mg of pork liver esterase (PLE-A). The evolution of the reaction is monitored by high performance liquid chromatography HPLC. After 9 min (38% conversion), the reaction mixture is added to 150 ml of HCCI ₃ chloroform. The layers are separated and the aqueous layer is lyophilized for solvent removal. The white residue from lyophilization is extracted 3 times with 10 ml of absolute ethanol. The extracts are filtered, combined and concentrated in vacuo to give 179 mg of crude oil. The crude material is then eluted on a 45 x 30 mm silica gel column, using a mixture of 3 x 75 ml of ethyl acetate and 5 parts of ethyl acetate with 1 part ethanol as eluent. The product (+) - FTC is isolated as a white solid (109 mg; 37% based on the initial butyrate). High-performance HPLC chromatographic analysis of compound (+) - FTC as a 5'-O-acetate derivative shows 97.4% enantiomeric excess; [a] O (20, D) + 113.4 ° (c = 2.53; absolute ethanol).

A similar reaction is carried out using 0.12 mmol (37 mg) of 5'-0-butyrate of FTC and 7 mg of PS-800 in 4.0 ml of 4 parts buffer solution pH = 8 and 1 part methylcyane CH ₃ CN. The reaction is considerably weaker than that with PLE-A pig liver esterase and requires 74 h for 59% conversion. Separated butyrate (11.4 mg; 31% of the initial amount) exhibits 94% enantiomeric excess, by high performance liquid chromatography HPLC.

Resolution of nucleoside enantiomers, with cytidine-deoxycytidine deaminase alternatively, cytidine-deoxycytidine deaminase is used to separate racemic mixtures of 2-hydroxymethyl-5- (cytosine-1-yl) -1,3-oxathiolane and derivatives of this compound, including 2 hydroxymethyl-5- (5-fluoro-cytosine-1-yl) -1,3-oxathiolane.

The enzyme catalyzes the deamination of the cytosine half in a uracil. It has been found that one of the 1,3-oxatiolan nucleoside enantiomers is the preferred substrate for cytidindesoxycytididamine. The enantiomer that is not converted to the uracil derivative (and is therefore still basic) is extracted from the solution with an acid solution. Care should be taken to avoid strongly acidic solutions (pH below 3.0), which may disrupt the oxathiolanic cycle. Cytidindesoxycytidinodezaminase can be isolated from rat liver or human liver, or can be expressed from recombinant sequences in a prokaryotic system, such as, for example, Escherechia Coli.

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The method of resolving the cytosine nucleoside enantiomers, using cytidine-deoxy-1 cytidinodezaminase, can be used as a single resolution method or can be used in combination with other methods of resolution, including the enzymatic hydrolysis resolution of 3 5'-O-nucleoside esters. , as described above.

The combination of enzymatic resolution and classical methods of resolution 5

The process described above for separating racemic mixtures of nucleoside enantiomers can be combined with other classical methods of enantiomeric separation, 7 for increasing the optical purity of the finished product.

The classical methods of separation include a variety of physical and chemical techniques. Often the most efficient and simplest technique is recrystallization, based on the principle that racemates are often much more soluble than the corresponding individual enantiomers. 11 Recrystallization can be carried out in any phase, including acylated compounds or the finished enantiomeric product. If the result is fully successful, this simple path is a 13 method worth choosing.

When recrystallization of a material with an acceptable optical purity is not achieved, other methods may be evaluated. If the nucleoside is basic (for example, a cytidine), chiral acids that form diastereomeric mixtures can be used which can have 17 significantly different solubility properties. Non-limiting examples of chiral acids include malic acid, mandelic acid, di-benzoyl tartaric acid, 3-bromcamfor-8-sulfonic acid, 19-10-camphorsulfonic acid and di-para-toluiltartric acid. Similarly, acylation of the free hydroxyl group with a chiral acid derivative thus results in the formation of diastereomeric mixtures whose physical properties may differ sufficiently to allow separation.

Small quantities of enantiomerically enriched nucleosides can be obtained or purified by treating the racemic mixture through an HPLC column that has been designated for chiral separations, including cyclodextrin-bonded columns, marketed by Rainin 25 Corporation.

Example 4. Separation of racemic mixtures of nucleosides by high performance liquid chromatography 27 HPLC

The resolutions of the C4 'enantiomers of (±) -FTC are performed using a chiral column with 29 cyclodextrin links (AC-I cyclolayer), obtained from Rainin Corporation (Woburn, MA). The conditions are as follows: 0.5% isocratic methanol, in water; fluid flow rate of 1 ml per 31 minutes, ultraviolet detection at 262 nm. HPLC purity methanol is obtained from J.T.Baker (Phillipsburg, N J). Racemic mixtures are injected and fractions are collected. Fractions containing each of the enantiomers mentioned are collected, frozen and then lyophilized. The compounds are characterized by ultraviolet spectroscopy and their 35 retention times by high performance liquid chromatography HPLC. In general (-) enantiomers have lower retention times than (+) enantiomers (see J. Liquid 37 Chromatography, 7: 353-376, 1984). The concentrations of the compounds are determined by ultraviolet spectroscopy, using a stock solution of known concentration (15 μΜ) 39 prepared in water for biological evaluation. The retention times for the separate enantiomers are presented in Table 2. 41

Table 2

Retention times of FTC compound enantiomers

compound R _f (minutes) (-) - FTC 8.3 (+) - FTC 8.7

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Example 5. Alternative methods for separating FTC enantiomers using a chiral column

Using a Cyclobond I-Ac (cyclicly bonded) column (5 pm, 25 cm x 4.6 ml, Rainin Corporation, Wobum, MA, Catalog No. AST-41049) with a fluid flow rate of 0.6 ml per minute of 0.5% isocratic methanol (Fisher Scientific, Inc. HPLC catalog no. A-452-4 in water) and ultraviolet detection at 262 nm, FTC enantiomers have retention times of 12.68 min ((-) FTC ) and 13.20 min ((+) - FTC).

Using a Chiralpak AS column (10 pm, 25 cm x 4.6 mm JTBaker Inc., PhilIsburg, NJ, catalog no. 7406-00, serial no. 09-29-10320) with a fluid flow rate of 0.8 ml per minute of isopropyl alcohol (grade for HPLC High Performance Liquid Chromatography, Fisher Scientific, Inc. Cat. No. A-451-4) and ultraviolet detection at 262 nm, FTC compound enantiomers have retention times of 5.9 min ((-) - FTC) and 9.8 minutes ((+) - FTC).

III. The ability of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) 1,3-oxathiolane (FTC) to inhibit HIV replication

It is often desirable to screen for a number of racemic mixtures of nucleosides as a preliminary phase to determine whether further separation into the enantiomerically enriched components and further evaluation of antiviral activity are warranted. Nucleoside capacity to inhibit human immunodeficiency virus HIV can be measured by various experimental techniques. The techniques used in the present invention, which are described in detail below, measure the inhibition of viral replication in phytohemagglutinin (PHA) in human peripheral blood mononuclear cells (PBM) stimulated with HIV-1-infected phytohemoglutinin (PHA) (LAV strain). The amount of virus produced is determined by measuring the reverse transcriptase enzyme on the encoded virus. The amount of enzyme produced is proportional to the amount of virus produced. Table 3 shows the EC ₅₀ values (nucleoside concentration that inhibits virus replication in 50% of stimulated human peripheral blood mononuclear cells (PBM) estimated with an error factor of 10%) and IC ₅₀ values (nucleoside concentration that inhibits 50% mononuclear cell development non-infected human mitogen-stimulated peripheral blood (PBM) of (±) -1,3-oxatiolan and nucleosides.

Example 6. Anti-HIV (±) -1,3-oxathiolane activity of nucleosides

A. Stimulated phytohemagglutinin PBM cells, 3 days old (10 ⁶ cells per ml) from seronegative healthy donors to HIV-1 and hepatitis B virus, are infected with HIV-1 (LAV strain) at a concentration about 100 times the dose that infects 50% tissue culture (TICD) per ml and these are grown in the presence and absence of different concentrations of antiviral compounds.

B. At about one hour after infection, the medium with the compound to be tested (2 times the final concentration in the medium) or without the compound, is added to the vials (5 ml, with the final volume of 10 ml). AZT is used as a positive control.

C. The cells are exposed to the virus (approximately 2 x 10 O ⁵ dpm / ml, as determined by reverse transcriptase analysis) and then placed in a CO ₂ incubator. HIV-1 (LAV strain) is obtained from the Centers for Disease Control in Atlanta, Georgia. The methods used for culturing PBM cells, harvesting the virus and determining the activity of reverse transcriptase are those described by Mc Dougal et al., (J. Immun. Meth. 76,171-183,1985) and Spira et al., (J. Clin. Meth. 25,97-99,1987) except that fungizone was not included in the environment (see Schinazi et al., Antimicrob. Agents Chemother. 32,1784-1787 (1988); Id 34: 1061-1067 (1990)).

D. On day 6, the cells and the supernatant are transferred to a 15 ml tube and centrifuged at approximately 900 g for 10 min. 5 ml of supernatant is removed and the virus is concentrated by centrifugation at 40,000 rotations per minute for 30 minutes (Beckman

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70.1 Ti rotor). The pellet with solubilized virus is processed to determine the levels of 1 reverse transcriptase. The results are expressed in dpm / ml of the supernatant sample. The virus in smaller volumes of supernatant (1 ml) can therefore be concentrated by centrifugation 3 before solubilization and determining the reverse transcriptase levels.

The effective median concentration (EC ₅₀ ) is determined by the median effect method 5 (Antimicrob. Agents Chemother. 30, 491-498 (1986)). In summary, the percentage of virus inhibition, as determined from reverse transcriptase measurements, is plotted against the micromolar concentration of the compound. EC ₅₀ is the concentration of the compound at which there is a 50% inhibition of viral development. 9

E. Mitogen-stimulated uninfected human PBM cells (3.8 x 10 ⁵ cells per ml) are cultured in the presence and absence of the drug under conditions similar to those used for the 11 antiviral assays described above. Cells are counted after 6 days, using a hemacytometer and exclusion method with Trypan Blue, as described by 13 Schinazi et al., Antimicrobial Agents and Chemotherapy22 (3), 499 (1982). The IC 50 value is the concentration of the compound that inhibits 50% of normal cell growth. 15

Table 3 17

EC ₅₀ and IC ₅₀ at different 1,3-oxatiolan nucleoside analogues in human PBM cells

Code X or Y R Antiviral cytotoxicity EC ₆₀ , μΜ Ιθ5ο> MM DLS-009 X = O H > 100 > 100 DLS-010 X = O Me 64.4 > 100 DLS-027 X = O F > 100 > 100 DLS-028 X = O BUT 60.8 > 100 DLS-044 X = O br > 100 > 100 DLS-029 X = O I > 100 > 100 DLS-020 y = nh ₂ H 0.02 > 100 DLS-011 y = nh ₂ Me > 10 > 100 DLS-022 y = nh ₂ F 0.1 > 100 DLS-023 y = nh ₂ BUT 38.7 > 100 DLS-021 y = nh ₂ br 77.4 > 100 DLS-026 y = nh ₂ I 0.72 > 100 DLS-058 (-) y = nh ₂ F 0,008 > 100 DLS-059 (+) y = nh ₂ F 0.84 > 100 DLS-053 y = nh ₂ cf ₃ 60.7 > 100

As shown in Table 3, generally, the substituted cytosine 1,3-oxathiolane 37 nucleosides are much more active than the corresponding uracil nucleosides. Errors in EC ₆₀ and IC ₅₀ measurements are estimated at + 10%. 39

One of the (±) -FTC compounds (referred to as compound 8 DLS 022) exhibits not only exceptional activity (about 10 nM in PBM cells), but also very low toxicity (> 100 pm in PBM, Vero and CEM cells ).

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IC _50s of (+) - FTC are above 100 µM, indicating that the compound is non-toxic in uninfected PBM cells, up to 100 µm.

or

Example 7. Antiviral activity of FTC compound enantiomers, separated by HPLC

The enantiomers of the FTC compound are isolated by the method of example 4 and the antiviral activity is evaluated by the method of example 6. The results are presented in table 4 and illustrated in fig. 5.

Table 4

Antiviral activity of (+) and (-) enantiomers of the FTC

Treatment concentration pM DPM / ml % inhibition (corrected) EC ₆₀ : μΜ FTC (+) 0.0001 73.755 26.6 0,018 0,005 83.005 16.3 0.01 60.465 41.3 0.05 34.120 70.4 0.1 14,160 92.4 0.5 18.095 88.1 1 7555 99.7 5 7940 99.3 10 5810 101.7 FTC (-) 0,001 76.275 23.8 0.02 0,005 58.590 43.3 0.01 75.350 24.8

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Table 4 (continued) 1

Treatment concentration μΜ DPM / ml % inhibition (corrected) ECso- μΜ 0.05 28.890 76.20 0.1 13.175 93.5 0.5 9485 97.6 FTC (+) 0,001 94.340 3.8 0.28 0,005 107.430 -10.6 0.01 99.465 -1.8 0.05 87.120 11.8 0.1 86.340 12.7 0.5 33.225 71.4

As shown in Table 4, in this experiment the enantiomer (-) of compound 13 FTC appears to be more active by about an order of magnitude than the enantiomer (+) of compound FTC and has approximately the same anti-HIV activity as the racemic mixture. . Neither 15 enantiomers nor racemic mixture were toxic, up to 100 μΜ, as measured by the Tryptan Blue exclusion method in human PBM cells. 17

Example 8. Antiviral activity of FTC enantiomers, separated by the method of example 3 19

The (±) -FTC enantiomers are thus separated by the method of example 3 and the antiviral activity is evaluated by the method of example 6. The results are illustrated in fig. 6. As shown in FIG. 6, EC ₅₀ of the racemic mixture of the FTC compound is 0.017 μ 0,0, EC ₅₀ of the (-) - FTC compound at 0.0077 μΜ and EC ₅₀ of the (+) - FTC compound at 0.84 pM. 2. 3

Example 9. Absorption (+) - FTC in human PBM cells

Studies were performed using radiolabelled FTC compound, on the following intracellular profiles of the source drug and on metabolites detected in cells. All studies were conducted in duplicate. Human peripheral blood mononuclear cells (27 PBM cells) were suspended in RPML1640, the medium containing 10% fetal calf serum and antibiotics (2 x 10 ⁶ cells per ml), 10 ml for the fixed time and incubated with 10 μΜ FTC ( activity 29 specific about 700 dpm / pmol) The cells are then exposed to the drug for 2,6,12 and 24 hours. At these fixed times, the medium is separated and the cells are washed twice 31 with a salt solution, cooled, Hank, balanced. The extraction is carried out with the addition of 0.2 ml of 60% methanol / cold water and kept until the following day at -70'C. The next 33 in the morning, the suspensions are centrifuged and the extractions are repeated twice, for 0.5 h at -70 ° C. Total supernatants (0.6 ml) are lyophilized until dry. The residues 35 are resuspended in 250 µl of water and the aliquots between 50 and 100 µl are analyzed by high performance liquid chromatography HPLC. Quantitative analysis of drug 37 of intracellular origin and metabolic derivatives is conducted by high performance liquid chromatography HPLC. Because some of these compounds have potential acid lability, the buffer system close to the physiological pH is used to separate the metabolites.

Fig. 7 is a graph of the presence (consumption) of tritium (±) -FTC compounds in cells 41

Human PBM (mean of two determinations) in time (hours) versus pmol / 10 ⁶ cells. Consumer studies indicate that the radiolabeled FTC compound is already absorbed in human lymphocytes, 43 which produce very large amounts of the 5'-triphosphate derivative of the FTC compound.

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Example 10. Antiretroviral activity of the FTC compound in different cell lines

The antiretroviral activity of the FTC compound is measured in a number of cell lines, using procedures similar but not identical to those mentioned in Example 6. The cell lines are obtained from either human donors or the AIDS Research and Reference Reagent Program , NIH, Rockville, Maryland, ATCC or Red Cross. Timid-deficient cells in CEM kinase are prepared by sequential passage of EMC cells in the presence of 5-bromo-2'-deoxyuridine. The results are presented in table 5.

Table 5

Antiretroviral activity of FTC in different cellular systems

Cellular system (virus strain) EC ₅₀ (μΜ (+) - FTC HIV-1 PBMC (LAV-1) 0.027 MT2 (HTLV ₁₁₁₈ ) 0.89 EMC (LAV-1) 0.08 CEM-TK ⁽⁾ (LAV-1) 0,026 CEM (HTLV _111B ) NTH 0.09 HIV-2 PBMC (ROD2) 0.0038 (±) FTC 0.0007 (-) FTC 0.026 (+) - FTC SIV AA-2 (SIV251) 4.6 C-8166 (SIV251) <8.0 IVF CrFK (61E) <1

Example 11. Exit of (±) -FTC compounds from human PBM cells

Studies using radiolabeled FTC were performed to track intracellular profiles of the source drug and metabolites detected in the cell after incubation in the drug medium for 24 hours, after which the drug is removed. This study measures the time required for intracellular levels of triphosphates to decrease. Studies are performed in duplicate. Uninfected cells (2 x 10 ⁶ ml) are suspended in a suitable medium supplemented with serum (10 ml for the fixed time) and incubated at 37 ° C in the 5% CO ₂ incubator. The concentration of the radiolabelled FTC compound is 10 μΜ. After pulsing the cells with the labeled compound for 24 hours, the cells are thoroughly washed and then filled with fresh medium without antiviral drugs (0 hours). At 0, 2, 4, 6.12, 24 and 48 h (secondary incubation time), the cells are removed and then immediately extracted with a 60% cold methanol / water mixture. Extracts are obtained by centrifugation and removal of cell pellets. The extracts are lyophilized and then stored at -70 ° C. Prior to analysis, the material is resuspended in 250 µl of HPLC buffer. Quantitative analysis of the drug of intracellular origin and metabolic derivatives is performed by high performance liquid chromatography FPLC, using either a Micrometrics or an HPLC 1090 Hewlett-Packard model,

EN 122814 Β1 with a column of Partisil 10 SAX anionic exchangers (Whatman Inc.) with a fluid flow rate of 1 ml per minute, at a pressure of 0.07 kg / cm ² (1 kpsi), with a detection ultraviolet at 262 nm. Neionozată water mobile phase consisted of (A), 2 mM NaH ₂ PO _4/16 mM NaOAc 3 (pH = 6.6) (B), 15 mM NaH ₂ PO ₄ / 120.2 mM NaOAc (pH = 6.6 ) (C), and 100 mM NaH ₂ P0 _4/800 mM NaOAc (pH = 6.6) (D). 5

Separation method: isocratic for 5 min with A, followed by linear gradient 15 min up to 100% B, followed by linear gradient 20 min up to 100% C, followed by linear gradient 10 min 7 up to 100% D, followed by isocratic 30 min with 100% D.

Retention times (minutes) in human cells

compound Unchanged monophosphate diphosphate triphosphate (±) FTC 5.0 39.0 55.0 68.0

Fig. 8 is a graphical representation of the output of (±) -FTC compounds labeled from human PBM cells, measured in hours, after drug withdrawal from concentration 15 (pmol / 10 ⁶ cells). As shown in this figure, FTC-triphosphate has an intracellular half-life of approximately 12 h and can be readily detected intracellularly at concentrations of 1-5 μΜ at 48 h after extracellular drug removal is the value above EC ₅₀ for this compound. Further, the affinity (K ¹ ) for (±) -FTC triphosphate 19 using HIV RT is 0.2 µM, which is below the concentration level at 48 h.

Example 12. Anti-HIV activity of pharmaceutically acceptable derivatives of compounds 21 (±) -FTC

a. The number of pharmaceutically acceptable derivatives of compound (±) -23

FTCs prepared by derivatizing positions 5'and N ⁴ are evaluated for anti-HIV activity in PBM cells, using a procedure similar to that described in Example 6. The results 25 are as follows. The 5'-0-butyrate ester of (±) -FTC compound has an EC ₅₀ value of 0.0017. The N ⁴ -acetyl derivative of compound (±) -FTC has an EC ₅₀ value of 0.0028.27

The ^4- ester 5'-0-butyrate of the (±) -FTC compound has an EC ₅₀ value of 0.0058.

b. The anti-HIV activity of 5'-0-butyrate ester of compound (±) -FTC in system 29 MT4 (EC _S0 ) is 0.04 μΜ. In the same analysis the unbound (±) -FTC compound exhibits an IC ₅₀ value of 0.52 pM. The IC ₅₀ value for AZT in this system is 0.09 pM.31

V. The ability of the FTC compound to inhibit HBV replication

Example 13. Evaluation of the activity of (+) and (-) atoms of FTC in cell cultures33

2.2.15

The ability of the FTC compound enantiomers to inhibit virus development in cell cultures 2.2.15 (HepG2 cells transformed with hepatitis virion) is described in detail below. A summary and description of the analysis for antiviral effects in this 37 culture system and the analysis of DNA deoxyribonucleic acid in hepatitis B virus (Korba and Milman, 1991, Antiviral Res., 15: 217) were described. Antiviral assays are performed on two separate 39-cell passages. All the wells in all the plates are sown at the same density and at the same time. 41

Analysis parameters. Due to the inherent variations in intracellular and extracellular DNA HBV levels, only depressions greater than 3.5 times (for deoxyribonucleic acid 43

HBV virion DNA) or 3.0-fold (for deoxyribonucleic acid DNA replication intermediates to HBV) from the mean levels for these forms of HBV DNA, which are formed in untreated cells, are considered to be significant. statistical. (P <0.05). Levels of deoxyribonucleic acid DNA integrated into hepatitis B virus HBV at each acid preparation 47

RO 122814 Β1 deoxyribonucleic cellular DNA (which remains constant, relative to a pair of cells, in these experiments) is used to calculate levels of intracellular DNA deoxyribonucleic acid forms of HBV, thus ensuring that equal amounts of acid have been compared between separate samples. deoxyribonucleic cellular DNA.

Typical values for extracellular HBV virion DNA deoxyribonucleic acid in untreated cells range from 50 to 150 µg / ml in culture medium (approximately 76 µg / ml on average). Deoxyribonucleic acid DNA replication intermediates for intracellular HBV virus in untreated cells range from 50 to 100 µg / µg of deoxyribonucleic acid cellular DNA (about 74 µg / µg of cellular DNA deoxyribonucleic acid). In general, decreases in the levels of deoxyribonucleic acid DNA of intracellular HBV virus, due to treatment with antiviral compounds, are less pronounced and occur much slower than decreases in levels of deoxyribonucleic acid DNA in the HBV virion (Korba and Milman, 1991, Antiviral Res., 15: 217).

The manner in which hybridization assays are performed for these experiments resulted in the equivalence of approximately 1.0 µg of intracellular deoxyribonucleic HBV DNA up to 2-3 genomic copies per cell and 1.0 µg / ml of deoxyribonucleic acid HBV DNA. extracellular up to 3 x 10 ⁵ viral particles per ml.

Toxicity analysis

Toxicity assays are performed to determine whether the observed antiviral effects are due to a general effect on cell viability. The method used in the present invention was the measurement of natural red color absorption, a standard and widely used assay for cell viability in a variety of host systems for viruses, including HSV and HIV. Toxicity analyzes are performed on flat tissue culture plates at the bottom with 96 wells. Cells for toxicity assays are cultured and treated with test compounds using the same scheme described below for antiviral assays. Each compound is tested at 4 concentrations, each in triplicate cultures (wells A, B and "C). The absorption of natural red dye is used to determine the relative level of toxicity. The dye absorbance internalized at 510 nm (A _sin ) is used for quantitative analysis. Values are presented as a percentage of the average A _sin values in 9 separate cultures of untreated cells maintained on the same 96 well plate as the test compounds. The absorption of the dye in the 9 control cultures on plate 5 is between 91.6 to 110.4% and on plate 6 from 96.6 to 109%. The results are given in table 6.

Table 6

Toxicity analysis of test compounds in cells 2.2.15

disc Compound Conc. (ΜΜ) Color absorption (% of control) Enjoy A Enjoy B Enjoy C 5 DMSO 10.0 * 0.7 1.6 0.9 3.3 55.9 68.7 61.7 1.0 91.2 96.4 106.8 0.3 98.7 102.9 93.5

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Table 6 (continued) 1

disc Compound Conc. (ΜΜ) Color absorption (% of control) Enjoy A Enjoy B Enjoy C 6 (-) - FTC 300 53.0 51.1 51.5 100 64.1 66.6 77.6 30 98.7 94.3 96.4 10 94.3 94.9 92.2 6 (+) - FTC 300 43.4 56.7 58.5 100 77.7 66.3 72.1 30 81.1 88.3 88.1 10 90.9 99.4 90.5

* For DMSO, concentrations are presented as percentages of original stock solution.

* For DMSO, concentrations are presented as percentages of original stock solution.

Toxicity assessment

As indicated in Table 6, no significant toxicity (greater than the 50% decrease in absorption levels observed in untreated cells) was observed for the test compounds at concentrations used for antiviral assays. Both of the 19 test compounds, (-) - FTC and (+) - FTC, appear to be toxic at higher concentrations used for toxicity tests (330 μΜ). 21

Antiviral assessments

Control 23 within normal variations, deoxyribonucleic acid DNA levels for the virion

HBV and HBV intracellular replication intermediates (HBV RI) remain constant in untreated 25 cells over the tested period. DMSO at a concentration of 1% does not affect the levels of HBV replication in cell cultures 2.2.15. 27

Test compounds

As shown in Table 7, both (-) - FTC and (+) - FTC compounds significantly inhibit HBV replication at test levels. As shown in Table 8, compound (-) - FTC still significantly inhibits DNA deoxyribonucleic acid synthesis in HBV virion and DNA deoxyribonucleic acid synthesis 31 in intracellular HBV at concentrations of 4.1 and 0.25 μΜ.

Table 7

Effect of test compounds on HBV production in cell cultures 2.2.15. 35

well Treatment DNA in HBV virion * (pg / ml culture medium) DNA at intracellular HBV (pg / pg cellular DNA) Day 0 Day 4 Day 9 MONO RI 7A Untreated cells 59 75 94 2.7 93 7B Untreated cells 47 64 88 2.5 93 8A Untreated cells 65 100 71 2.2 97 8B Untreated cells 77 65 110 2.4 62

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Table 7 (continued)

well Treatment DNA in HBV virion * (pg / ml culture medium) DNA at intracellular HBV (pg / pg cellular DNA) Day 0 Day 4 Day 9 MONO R 7K DMSO @ 1.00% 100 50 48 1.9 95 7L DMSO @ 1.00% 48 96 54 2.8 98 8K DMSO @ 1.00% 93 63 68 2.2 86 8L DMSO @ 1.00% 66 57 59 1.6 97 9U (-) - FTC @ 10 μΜ 120 36 1 1.1 14 9V (-) - FTC @ 10 μΜ 89 48 1 1.5 19 10U (-) - FTC @ 10 μΜ 58 41 0.1 1.9 13 10V (-) - FTC @ 10 μΜ 110 32 0.1 1.2 16 9W (+) - FTC @ 10 pM 88 42 0.1 0.8 14 9X (+) - FTC @ 10 μΜ 58 57 0.2 0.4 19 10W (+) - FTC @ 10 μΜ 69 55 0.1 0.7 17 10X (+) - FTC @ 10 μΜ 45 39 0.1 0.4 15

The DNA separation sensitivity for HBV virion is 0.1 µg / ml.

@ Intracellular HBV DNA was analyzed 24 h, following the 9th day of treatment. The levels of HBV DNA integrated into each cell DNA preparation are used to calculate the levels of 3.2 kb episodic HBV genomes (MONO.) And deoxyribonucleic acid DNA replication intermediates (Rl) in the case of HBV virus.

Table 8

Effect of test compounds on HBV production in cell cultures 2.2.15.

well T reatment DNA in HBV virion * (pg / ml culture medium) DNA at intracellular HBV * (pg / pg cellular DNA) Day 0 Day 4 Day 9 MONO. R 31A Untreated cells 64 54 65 2.8 65 31B Untreated cells 51 54 77 2.0 53 32A Untreated cells 100 76 56 3.5 81 32B Untreated cells 53 97 83 3.1 68 35A (-) - FTC @ 4 μΜ 74 27 > 0.1 1.4 1 35B (-) - FTC @ 4pm 87 28 > 0.1 0.5 1 36A (-) - FTC @ 4 μΜ 120 20 1 0.9 1 36B- (-) - FTC @ 4 μΜ 59 16 0.2 0.2 2 35C (-) - FTC @ 1 μΜ 70 13 > 0.1 1.7 2

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Table 8 (continued) 1

well Treatment DNA in HBV virion * (pg / ml culture medium) DNA at intracellular HBV * (pg / pg cellular DNA) Day 0 Day 4 Day 9 MONO. R 35D (-) - FTC @ 1 μΜ 62 15 > 0.1 1.2 3 36C (-) - FTC @ 1 μΜ 60 22 1 1.4 2 36D (-) - FTC @ 1 μΜ 89 28 0.3 1.5 4 35E (-) - FTC @ 0.25 μΜ 84 15 > 0.1 1.5 4 35F (-) - FTC @ 0.25 μΜ 89 16 4 2.2 4 36E (-) - FTC @ 0.25 μΜ 66 13 1 1.8 8 36F (-) - FTC @ 0.25 μΜ 49 19 0.1 0.3 9

The separation sensitivity for deoxyribonucleic acid DNA to the HBV virion is 0.1 µg / ml.

The separation sensitivity for deoxyribonucleic acid DNA to the HBV virion is 0.1 µg / ml.

@ Intracellular HBV DNA analysis was at 24 h, following the 9th day of treatment. The levels of HBV DNA integrated into each cellular DNA preparation are used to calculate 15 levels of 3.2 kb episodic HBV genomes (MONO) and deoxyribonucleic acid DNA replication intermediates (Rl) in the case of HBV virus. 17

Example 14. Absorption (+) - FTC in human liver cells: HBV activity on FTC compound 19

The procedure of Example 9 is repeated with cells from the human liver (HepG2 cells, available from ATCC) to determine the uptake and metabolism of FTC 21 in these cells. As shown in FIG. 9, compound (±) - FTC is absorbed by HepG2 cells in large quantities. These cells in the human liver metabolize a high percentage of (+) - FTC to (±) -FTC triphosphate.

These data, in conjunction with other data provided in the present invention, show that (±) - 25

FTC as well as the (-) and (+) enantiomers of this compound, are phosphorylated in liver cells. These cells can be transformed with hepatitis B. virus 27

Example 15. Exit of the FTC compound from human HepG2 cells

Fig. 10 illustrates the release of compound [ ³ H] - (±) -FTC and its phosphorylated derivatives 29 in human HepG2 in pmol / 10 ⁶ cells over time, after pulsing cells with 10 μΜ [ ³ H] - (±) -FTC (700 DPM / pmol) for 24 h and evaluating the concentration of the compound at 24 h after separation. 31 Fig. 11 illustrates the combined decrease in [ ³ H] - (±) -FTC and phosphorylated derivatives of this compound from human HepG2 cells after incubation with 10 μΜ 33 [ ³ H] - (±) -FTC (700 DPM / pmol ) for 24 h in pmol / 10 ⁶ cells over time.

As illustrated, even at 48 h, more than 1 μΜ of the active compound (which is 35 significantly higher than EC ₅₀ for the compound) is still present in the cells.

V. Toxicity in granulocyte-macrophage precursor cells 37

Example 16. Effect of FTC on granulocyte-macrophage precursor cell colony formation 39

Fig. 12 is a graphical representation of the effect of the (+) and (-) enantiomers of the compound

FTC on granulocyte-macrophage precursor cell colony formation, as measured in survival percentages relative to concentration in μΜ of (-) - FTC in open circle; (+) - FTC in closed circle; AZT closed square. As indicated, the (-) enantiomer of 43 FTC appears to be less toxic, for example it has a higher value for IC ₅₀ , than either the (+) or AZT enantiomer in this cell line. 45

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VI. Pharmacokinetics of FTC compound

Example 17. Metabolism of FTC compound in rats

Compound (±) -FTC is administered intravenously at doses of 10, 50 and 100 mg per kilogram in rats. The surface area under the curve was evaluated for plasma drug concentration versus time (AUC), total clearance (CL _Ț ), steady-state volume of distribution (V _ss ), mean stay time (MRT) and half-life (t _{1 / 2} ). The results are presented in table 9.

Table 9

Pharmacokinetic parameters of FTC after intravenous administration of 10, 50,100 mg per kilogram in rats

Dose AUC 1— o VSS MRT L / 2 mg / kg mg h / L L / h / kg L / kg h h 10 9.65 0.988 0.758 0.768 0.757 50 57.11 0.874 .699 0,800 0.815 100 120.72 0.830 0.663 0.798 0.969

* AUC = surface area under drug concentration curve in plasma relative to time; CL = total release; Vss = volume in steady state of the distribution; MRT = clearence and t _V2 = half-life.

Example 18. Pharmacokinetic parameters for FTC after oral and intravenous administration of FTC

Independent model pharmacokinetic parameters are derived for (±) -FTC by (intravenous (i.v.) and oral (p.o.)) administration of 33.3 mg / kg in Rhesus monkeys. The results are presented in table 10. It is important that the average bioavailability of the compound in monkeys is 73% (± 6%).

Table 10

Independent model pharmacokinetic parameters derived for FTC after intravenous (i.v.) or oral (p.o.) administration of 33.3 mg / kg in Rhesus monkeys *

The monkey AUC mg h / L CL _T L / h / kg v _5S L / kg <: -1 tl / 2 h K _a h ¹ F % I.V. Ruh 19.14 1.74 2.71 1.56 1.28 RMI 26.31 1.26 1.97 1.56 1.22 RJD 22.51 1.48 2.00 1.36 1.47 Mediate 22.65 1.49 2.23 1.49 1.32 ± S.D.. 3.59 0.24 0.42 0.12 0.13 P.O. Ruh 13.21 2.07 1.58 0.48 71 RMI 21.11 2.32 1.08 0.43 80 RJD 15.29 3.23 1.47 0.31 68 Mediate 16.54 2.54 1.38 0.41 73.00 (± 6) S.D. 4.09 0.61 0.26 0.09 6.24

* AUC = the surface below the plasma drug concentration curve over time; CL = total release; Vss = volume in steady state of the distribution; MRT = average dwell time and t _1/2 = half-life; F = bioavailability; and K _a = first order constant of the absorption speed.

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Table 11 1

The CSF / serum ratio of the FTC compound and the deaminated metabolite of this compound at one hour after treatment_________________________ 3

The monkey Path FTC Metabolite (FTU) RUH I.V. 0.076 0.024 RMI I.V. 0.062 0.032 RJD I.V. 0.162 0.052 Mediate 0,100 0.036 ± S.D.. 0.054 0.014 RUH P.O. 0.048 0,026 RMI P.O. 0.039 0.037 RJD P.O. 0,117 0.055 Mediate 0.068 0.039 ± S.D.. 0.043 0,015

Example 19. CSF / serum ratio of FTC compound and its metabolites in Rhesus monkeys. 17

The ability of (±) -FTC compounds to cross the blood-brain barrier was assessed by administering 33.3 mg per kilogram of the active compound to Rhesus 19 monkeys and by measuring the amount of (±) -FTC compound in cerebrospinal fluid (CSF). and in the bloodstream one hour after administration. The results are presented in Table 11. The data show 21 that a significant amount of the active compound crosses the blood-brain barrier in this mammal. 2. 3

ARE YOU COMING. Preparation of pharmaceutical compositions

People suffering from diseases caused by an infection with the HBV hepatitis B virus or with the HIV immunodeficiency virus can be treated by administering to the patient an effective amount of the (±) -FTC compound or the (-) or (+) enantiomers of this compound, or from a derivative or salt of this pharmaceutically acceptable compound, in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any convenient means, such as oral, parenteral, intravenous, intradermal, subcutaneous or topical, in liquid or solid form. 31

The active compound is included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to the patient a therapeutically effective amount 33 of the compound that inhibits viral replication in vivo, particularly in the case of HIV virus replication. and HBV, without causing serious toxic effects to the treated patient. Inhibitory amount is meant the amount of active ingredient which is sufficient to exert an inhibitory effect, as measured, for example, by the analysis performed as mentioned above.

A preferred dose of (-), (+) or (+) - FTC, for all the conditions mentioned above 39, will be in the range of about 1 to 50 mg per kilogram, preferably 1 to 20 mg per kilogram. body weight per day, more generally 0.1 to about 41 mg 100 mg per kilogram body weight of the body receiving the dose, per day.

The effective dosage range of pharmaceutically acceptable derivatives can be calculated based on the weight of the nucleoside of origin to be released. If the derivative itself has activity, the actual dosage can be estimated as above, using the weight of the derivative or by other means known in the literature.

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The compound is conventionally administered in units of any convenient dosage form, including, but not limited to, from 7 to 3000 mg, preferably from 70 to 1400 mg of active ingredient per unit of dosage form. unit. An oral dosage of 50 to 1000 mg is usually convenient. Ideally, the active ingredient will be administered so as to obtain the peak concentration of the active compound in plasma from about 0.2 to 70 μΜ, preferably from about 1.0 to 10 μΜ. This can be achieved, for example, by intravenous injection of a 0.1 to 5% solution of the active compound, optionally in saline, or administered as a bolus of active ingredient.

The concentration of active compound in the drug composition will depend on the rates of absorption, inactivation and excretion of the drug, as well as other factors known in the literature. It should be noted that the dosage values will also vary with the degree of severity of the condition that needs to be tanned. It should be further understood that for any particular subject, the specific dosage regimes should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the compositions and that the dosing ranges set forth in the present invention be given only as examples. , without intending to limit the purpose or practice of the claimed composition. The active ingredient may be administered at once or may be divided into a number of smaller doses to be administered at different time intervals. A preferred route of administration of the active compound is the oral route. Pharmaceutical compositions that can be administered orally will generally include an inert diluent or an edible vehicle. These compositions may be included in gelatin capsules or may be compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with the excipients and used in the form of tablets, pills or capsules. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, pills and other similar forms may contain any of the following ingredients or compounds of a similar nature: a binder, such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel or corn starch; a lubricant such as magnesium stearate or Steroids; a glider such as, for example, colloidal silicon dioxide; a sweetening agent such as sucrose for example sucrose or a flavoring agent, such as peppermint, methyl salicylate or orange flavorings. When the dosage unit form is the capsule, it may contain in addition to the type of materials mentioned above, a liquid vehicle such as a greasy oil. In addition, unit dosage forms may contain various other materials that alter the physical form of the dosage unit, such as sugar coatings, shredding shell, or other substances acting as enteric agents.

(±) -FTC or its (-) or (+) enantiomers or derivatives or pharmaceutically acceptable salts thereof may be administered as a constituent of elixir, suspension, syrup, pickle, chewing gum or other similar forms. A syrup may contain in addition to the active compounds, sucrose or a sweetening agent and certain preservatives, dyes, paints and flavoring substances.

(±) -FTC or enantiomers (-) or (+) or pharmaceutically acceptable derivatives or salts of this compound may also be mixed with other active materials that do not interfere with the desired activity or with other materials that supplement the desired action. , such as antibiotics, antifungal, anti-inflammatory or other antiviral substances, including other anti-HIV nucleoside compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application may include the following components: a sterile diluent such as water for injections, saline, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as, for example, benzyl alcohol or methyl

RO 122814 Β1 parabens; anti-oxidants such as ascorbic acid or sodium bisulfate; chelating agents such as, for example, ethylenediaminetetraacetic acid, buffering agents such as acetates, citrates or phosphates, tonicating agents such as sodium chloride or dextrose. 3 The original preparation may be included in ampoules, disposable syringes or multi-dose vials, made of glass or plastic. 5

If intravenous, vehicles of physiological saline or phosphate buffered saline (PBS) are preferred. In a preferred embodiment, the active compounds 7 are prepared with vehicles that will protect the compound from rapid elimination from the body, such as delivering a controlled release formulation, including microencapsulated implants and delivery systems 9. Biocompatible, biodegradable polymers can also be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortho-11 esters and polylactic acid. The methods for preparing these formulations are obvious to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and 13

Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeting cells infected with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable vehicles. These suspensions may be prepared according to methods 17 known to those skilled in the art, such as those described in US Pat. No. 4,228,811 (which are fully incorporated by reference in the present invention). For example, liposome formulations can be prepared by dissolving appropriate lipids (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoil phosphatidyl choline 21, and cholesterol) in an inorganic solvent which is then evaporated and then left behind. thin thread of dry lipid on the surface of the container. An aqueous solution of the active compound 23 or its monophosphate, diphosphate and / or triphosphate derivatives are then introduced into the container. The container is then manually twisted to release the lipid material from the sides 25 of the container and to disperse the lipid aggregates, thus obtaining a liposomal suspension. 27

The monophosphate, diphosphate and triphosphate derivatives of the FTC compound can be prepared, as described below. 29

Monophosphate can be prepared according to the process given by Imai and co

J. Org. Chem. 34 (6), 1547-1550 (June 1969). For example, about 100 mg of 31 FTC compound and about 280 µl of phosphoryl chloride are stirred in about 8 ml of dry ethyl acetate at about 0 ° C for about four hours. 33 The reaction is stopped with ice. The aqueous phase is purified on an active carbon column, and the elution is carried out with 5% ammonium hydroxide in a mixture of equal parts of ethanol 35 and water. Evaporation of the eluent gives FTC-5'-ammonium monophosphate.

The diphosphate can be prepared according to the procedure described by Davisson and 37 collaborators in J. Org. Chem. 52 (9), 1794-1801 (1987). The diphosphate of the FTC can be prepared from the corresponding tosylate, which can be prepared, for example, by reacting 39 nucleoside with tosyl chloride in pyridine at room temperature for about 24 hours, then processing the product in an ordinary manner (for example by washing, drying and crystallization of the product).

Triphosphate can be prepared according to the procedure of Hoard and co., From J. Am. 43

Chem. Shock. 87 (8), 1785-1788 (1965). The FTC compound is activated (being converted to imidazolid according to methods known in the literature) and treated with tributyl 45 ammonium pyrophosphate in DMF dimethylformamide. The reaction primarily leads to the formation of nucleoside triphosphate, together with small amounts of unreacted monophosphate and diphosphate. 47

Purification by anion exchange chromatography on a DEAE column is followed by triphosphate isolation, such as tetrasodium salt. 49

Claims (15)

claims 1. Process for separating 1,3-oxathiolane derivatives, with the general chemical formula: NHRg characterized in that the racemic mixture of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3oxathiolane is subjected to a separation step under the action of an enzyme selected from the group consisting of esterase, lipase, substilisin, α- chymotrypsin, cytidine-deoxycytidine deaminase, which preferably catalyzes a reaction in one of the enantiomers, followed by exposure of the resultant product to the action of a second enzyme that intensifies the separation, recrystallization of the separation product and treating it with a chiral acid selected from a group consisting of malic acid, mandelic acid, 10-camphorsulfonic acid, 3-bromocampor-8-sulfonic acid, dibenzooyl tartaric acid and di-para-toluoylartartic acid. 2. Process according to claim 1, characterized in that the preparation of 1,3-oxathiolane derivatives comprises the steps of: (i) ozonation of a compound of formula CH ₂ = CH-CH ₂ -OR or RO-CH ₂ -CH = CH-CH ₂ OR, wherein R is a protecting group, yielding the compound of formula: wherein R _1a is a hydroxyl group protecting group; (ii) the cyclization reaction of the aldehyde obtained in step (i) with mercaptoacetic acid, when a lactone of the formula is obtained: (iii) reduction of the lactone obtained in step (ii) and its reaction with the carboxylic acid anhydride, yielding the compound of the formula: wherein L is a cleavable group; (Iv) optionally 5-fluorocytosine silylation with the protected amino group, yielding the compound: where R _1b is an amino protecting group, Z = Si (CH ₃ ) ₃ ; (v) coupling the silylated 5-fluorocytosine with the protected lactol to obtain the compound of the formula: wherein R _1a is a hydroxyl protecting group and R _1b is an amino protecting group, then the 19 protecting groups are optionally removed, giving 2 hydroxymethyl-5- (5-fluorocytosin1-yl) -1,3-oxathiolane. 21 Process according to claim 1, characterized in that the preparation of 1,3-oxathiolane derivatives can be carried out starting from the compound of formula: 23 wherein R _1a is a hydroxyl protecting group and R _1b is an amino protecting group. which react with a fluorination agent to introduce the fluorine atom into position 5 of the cytosine cycle, followed by the removal of the protecting groups. 4. The process according to claim 1, characterized in that the preparation of 1,3-oxathiolane derivatives can be carried out starting from the compound of the formula: wherein R _1a is a hydroxyl protecting group, which reacts with an agent that converts the oxo group at position 4 of the uracil nucleus into an amino group, followed by the removal of the protecting groups. Process according to claim 1, characterized in that the carboxylic acid anhydride is acetic anhydride. RO 122814 Β1 6. Process according to claim 1, characterized in that the esterase is the esterase from pig liver. Process according to claim 1, characterized in that the lipase is selected from a group consisting of the lipase from the pig pancreas and the lipase from Pseudomonas Cepacia. Process according to claim 1, characterized in that the racemic mixture is acylated at the C 5'-hydroxyl position, prior to the separation step, with a compound selected from a group consisting of substituted alkyl carboxylic acids and alkyl carboxylic acids. 9. Process according to claims 1 and 8, characterized in that the carboxylic alkyl acid is selected from the group consisting of acetic acid, propionic acid, butyric acid, pentanoic acid, 2-chloropropionic acid, 2-chlorobutyric acid and 2-chloropentanoic acid. The process of claim 1, characterized in that the racemic mixture is passed through a column including the enzyme immobilized on the carrier. Process according to claim 1, characterized in that the racemic mixture is mixed with the enzyme in a solution. Process according to Claim 1, characterized in that the enzymatic reaction takes place in the presence of a non-ionic surfactant, which is 4- (C ₈ H ₁₇ ) C ₆ H ₄ (OCH ₂ CH ₂ ) _n OH, with n ~ 10. Process according to claims 1-12, characterized in that it further comprises the step of separating the enantiomeric mixture, improved by a chiral column. Process according to claim 13, characterized in that the chiral column is a Cyclobond I-Ac column. Process according to claim 13, characterized in that the chiral column is a Chiralpak AS column.